Cadoni, E. | Search bioRxiv

bioRxiv2025

Non-enzymatic isothermal strand displacement and amplification (NISDA) does not enable sensitive nucleic acid quantification.

Enzyme-free isothermal amplification methods offer a promising alternative to enzymatic assays for nucleic acid detection, particularly in low-resource settings. The nonenzymatic isothermal strand displacement and amplification (NISDA) assay was recently introduced as a highly sensitive, enzyme-free detection strategy. Here, we attempted to replicate its reported performance. Despite extensive testing, we failed to replicate the reported sensitivity and could only produce detectable signals at extremely high target concentrations ([≥]1 x 1011 copies/{micro}L). Our results highlight the critical importance of independent validation in the development of nonenzymatic diagnostic assays.

molecular biology↗

bioRxiv2024

Individual G-quadruplex targeting with ligand-functionalized CRISPR-Cas9 uncovers transcriptional-dependent functional responses

The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, severely limiting their scope. Herein, we describe a new Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrated that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA revealed that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4-site. We further demonstrated that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides an innovative platform to investigate G4- and iM-biology with high precision and unveils the therapeutic relevance of individual DNA structures with unprecedented selectivity.

cell biology↗